The present invention relates to a human RNase III, the gene for which has now been cloned and characterized, and compositions and uses thereof. Antisense inhibitors of human RNase III are also described.
Ribonuclease III (RNase III) is an endoribonuclease that cleaves double stranded RNA. The enzyme is expressed in many organisms and is highly conserved. I. S. Mian, Nucleic Acids Res., 1997, 25, 3187-95. All RNase III species cloned to date contain an RNase III signature sequence and vary in size from 25 to 50 kDa. Multiple functions have been ascribed to RNase In both E. coli and S. cerevisiae, RNase III has been reported to be involved in the processing of pre-ribosomal RNA (pre-rRNA). Elela et al., Cell, 1996, 85, 115-24. RNase III has also been reported to be involved in the processing of small molecular weight nuclear RNAs (snRNAs) and small molecular weight nucleolar RNAs (snoRNAs) in S. cerevisiae. Chanfreau et al., Genes Dev. 1996, 11, 2741-51; Qu et al., Mol. Cell. Biol. 1996, 19, 1144-58. In E. coli, RNase III has also been reported to be involved in the degradation of some mRNA species. D. Court, in Control of messenger RNA stability, 1993, Academic Press, Inc, pp. 71-116.
A human double strand RNase (dsRNAse) activity has been described. Wu et al., J. Biol. Chem., 1998, 273, 2532-2542; Crooke, U.S. Pat. No. 5,898,031; U.S. Pat. No. 6,017,094. By the rational design and testing of chemically modified antisense oligonucleotides that contained oligoribonucleotide stretches of varying length, a dsRNAse activity was demonstrated in human T24 bladder carcinoma cells which produced 5xe2x80x2-phosphate and 3xe2x80x2-hydroxyl termini upon cleavage of the complementary cellular RNA target. This pattern of cleavage products is a feature of E. coli RNase III. The cleavage activity in human cells required the formation of a dsRNA region in the oligonucleotide. This human dsRNAse activity is believed to be useful as an alternative terminating mechanism to RNase H for antisense therapeutics. Because it relies on xe2x80x9cRNA-likexe2x80x9d oligonucleotides, which generally have higher potency than the xe2x80x9cDNA-likexe2x80x9d oligonucleotides required for RNase H activity, it may prove an attractive alternative to RNase H-based antisense approaches.
RNA interference (RNAi) is a form of sequence-specific, post-transcriptional gene silencing in animals and plants, elicited by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene. Elbashir et al., Nature, 2001, 411, 494-498. dsRNA triggers the specific degradation of homologous RNAs, only within the region of homology. The dsRNA is processed to 21- to 23-nucleotide fragments, sometimes called short interfering RNAs (siRNAs) which are believed to be the guide fragments for sequence-specific mRNA degradation. The processing of longer dsRNA to these short siRNA fragments is believed to be accomplished by RNase III. Elbashir et al., ibid., Elbashir, et al., Genes and Devel., 2001, 15, 188-200. Thus it is believed that the human RNase III of the present invention may be useful in further understanding and exploiting the RNAi mechanism, particularly in human cells.
Despite the substantial information about members of the RNase III family and the cloning of genes encoding proteins with RNAse III activity from a number of lower organisms (E. coli, yeast and others), no human RNase III has previously been cloned. This has hampered efforts to understand the structure of the enzyme(s), its distribution and the functions it may serve. The present application describes the cloning and characterization of a cDNA that expresses a human RNase III. Cloning and sequencing of the cDNA encoding human RNAse III allowed characterization of the this nucleic acid as well as of the location and function of the RNAse III protein itself.
The present invention provides a polynucleotide sequence (set forth herein as SEQ ID NO: 1) which has been identified as encoding human RNase III by the homology of the calculated expressed polypeptide (provided herein as SEQ ID NO: 2) with known amino acid sequences of yeast and worm RNase III as well as by functional analysis.
The present invention provides polynucleotides that encode human RNase III, the human RNAse III polypeptide, vectors comprising nucleic acids encoding human RNase III, host cells containing such vectors, antibodies targeted to human RNase III, nucleic acid probes capable of hybridizing to a nucleic acid encoding a human RNase III polypeptide, and antisense inhibitors of RNAse III expression. Methods of inhibiting RNase III expression or activity are also provided, as are pharmaceutical compositions which include a human RNase III polypeptide, an antisense inhibitor of RNAse III expression, or a vector containing a nucleic acid encoding human RNase III.
Methods for identifying agents which modulate activity and/or levels of human RNase III are also provided. Methods of promoting inhibition of expression of a selected protein via antisense, methods of screening oligonucleotides to identify active antisense oligonucleotides against a particular target, methods of prognosticating efficacy of antisense therapy, methods of promoting RNA interference (RNAi) in a cell and methods of eliciting cleavage of a selected cellular RNA target are also provided. All of these methods exploit the RNA-cleaving activity of RNase III. In preferred embodiments the oligonucleotides used in these methods are RNA-like oligonucleotides. Also provided are methods of identifying agents which increase or decrease activity or levels of human RNase III.
The polynucleotides, antisense oligonucleotides, polypeptides and other compounds, compositions and methods of the present invention are useful for research, biological and clinical purposes. For example, the polynucleotides and antisense oligonucleotides are useful in defining the roles of RNase III and the interaction of human RNase III and cellular RNA (including pre-mRNA or pre-rRNA).